Following our 1997 announcement of the successful production of the rhesus monkey infants, NETI and DITTO, by nuclear transfer from embryonic blastomeres, we have focused efforts on strengthening the infrastructure required for routine embryo culture, storage and development, evaluated twinning as a method for producing genetically identical animals and continued nuclear transfer studies. Our goals are to improve the nuclear transfer technology in non-human primates such that it can be used in the propagation of existing disease models or genetically valuable monkeys, in establishing a paradigm combining gene targeting with nuclear transfer to create new disease models and in efforts to develop HIV vaccines by making genetically identical animals available to the vaccine development community. In the area of infrastructure development, we have monitored oocyte performance by setting aside a cohort (5-7 Metaphase II oocytes) from each oocyte retrieval (N = 55) for fertilization by intracytoplasmic sperm injection (ICSI). ICSI is a convenient and predictable alternative to conventional insemination, which in the rhesus monkey requires sperm activation by exposure to dibutyryl cyclic AMP and caffeine. This activation step can introduce significant sperm agglutination. Up to 25% of the oocyte cohorts have been retrospectively categorized as blastocyst incompetent, that is, no embryos developed to the blastocyst stage following co-culture on buffalo rat liver cells in CMRL 1066 medium in the presence of serum. Unfortunately, we have no morphological correlates that allow the prospective exclusion of blastocyst incompetent oocyte cohorts from experimental studies. However, at least a retrospective refinement of results can now be undertaken based on these observations and the application of a quality control screen on each oocyte cohort. We have also evaluated a number of alternatives to co-culture, including the use of sequential media commonly employed in human embryology laboratories, without defining a system that consistently outperforms co-culture. The poorly defined nature of this co-culture system along with the labor-intensive activity of maintaining cells for use in co-culture are recognized as limiting factors. Approximately 60% of ICSIderived embryos in blastocyst competent cohorts develop to the blastocyst stage representing a "gold" standard for comparative purposes. Given the ability to culture ICSI-derived embryos to the blastocyst stage routinely, we have generated a renewed interest in non-surgical methods of transferring uterine stage embryos into the uterus. This approach is complicated by the long, tortuous nature of the cervix in the rhesus macaque. However, prescreened, multiparous animals can be used to this end. We have now established one pregnancy in 8 trials, with a live birth in August of 1999, following the non-surgical, transcervical replacement of ICSI-produced blastocysts. In twinning, we have concentrated on blastomere separation at the 2- or 4-cell stage following fertilization by ICSI. The technique involves micromanipulation in divalent cation-free medium. Demiembryos, produced by this technique and placed in culture, develop to expanded blastocysts at the same frequency as their intact counterparts and several pregnancies have resulted from the transfer of demi-embryo twin pairs into synchronized recipient females. One singleton birth has occurred and the first set of twins was lost spontaneously at 75 days of gestation due to an incompetent cervix. The major concern with this technology is efficiency, given a 30-50% pregnancy rate and a 30% twinning rate, the overall efficiency may not be high enough to be cost effective. Studies involving nuclear transfer have focussed on enucleation, fusion and chemical activation efficiencies. In the latter case, the in vitro development of parthenotes was monitored following exposure to ionomycin-DNAP or to the combination of cycloheximide and cytochalasin B. In both cases, parthenotes grew to the blastocyst stage with the same efficiency as ICSI-produced embryos allowing the conclusion that the chemical activation stimulus employed in NT is compatible with development. While we have experience with producing nuclear transfer (NT) embryos from fetal fibroblasts, their developmental potential in vitro at less than 10% has been disappointing. This has forced us to return to embryonic blastomere cloning and protocols are now available that support the development of NT embryos to the blastocyst stage at a 39% efficiency. Because of this relatively high efficiency, we have resumed the transfer of NT embryos to host mothers with the intent of establishing pregnancies.
|Original language||English (US)|
|Number of pages||2|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology